Carbon fixation systems and methods

ABSTRACT

Systems and methods for fixing carbon using bacteria are described. In one embodiment, a system includes a reactor chamber with a solution contained therein. The solution may include hydrogen (H2), carbon dioxide (CO2), bioavailable nitrogen, and a chemolithoautotrophic bacteria. The system may also include a pair of electrodes that split water contained within the solution to form the hydrogen. Additionally, the system may be operated so that a concentration of the bioavailable nitrogen in the solution is below a threshold nitrogen concentration to cause the chemolithoautotrophic bacteria to produce a product.

This Application is a Divisional of patent application Ser. No.15/758,843, filed Mar. 9, 2018, which is a national stage filing under35 U.S.C. 371 of International Patent Application Serial No.PCT/US2016/051621, filed Sep. 14, 2016, which claims the benefit ofProvisional Application Ser. No. 62/218,131, filed Sep. 14, 2015. Theentire contents of each of which are incorporated herein by reference intheir entirety.

GOVERNMENT FUNDING

This invention was made with government support under N00014-11-1-0725awarded by the Office of Naval Research and under FA9550-09-1-0689awarded by The Air Force Office of Scientific Research. The governmenthas certain rights in the invention.

FIELD

Disclosed embodiments are related to carbon fixation systems andmethods.

BACKGROUND

Sunlight and its renewable counterparts are abundant energy sources thatmay be harnessed to further sustainable production of materials. Forexample, photosynthetic organisms harness solar radiation to synthesizeenergy-rich organic molecules from water and CO₂. However, numerousenergy conversion bottlenecks exist in natural systems that limit theoverall efficiency of photosynthesis. Specifically, most plants do notexceed 1% conversion efficiencies and microalgae grown in bioreactors donot exceed 3% conversion efficiencies. However, conversion efficienciesof 4% for plants and 5%-7% for microalgae present in bubble bioreactorsmay be achieved in the rapid (short term) growth phase, but not overlonger periods. Additionally, although it is possible for artificialphotosynthetic solar-to-fuels cycles to have higher intrinsicefficiencies, they typically terminate at hydrogen production, with noprocess included to complete the cycle by carbon-fixation to creatematerials with higher energy densities.

SUMMARY

In one embodiment, a method includes: splitting water in a solutioncontaining a chemolithoautotrophic bacteria to form hydrogen (H₂) andoxygen (O₂) in the solution; providing carbon dioxide (CO₂) in thesolution; and limiting bioavailable nitrogen in the solution to below athreshold to cause the chemolitoautrophic bacteria to produce a product.

In another embodiment, a system includes a reactor chamber with asolution contained therein. The solution includes hydrogen (H₂), carbondioxide (CO₂), bioavailable nitrogen, and a chemolithoautotrophicbacteria. The system also includes a pair of electrodes that split watercontained within the solution to produce the hydrogen. A concentrationof the bioavailable nitrogen in the solution is below a thresholdnitrogen concentration to cause the chemolitoautrophic bacteria toproduce a product.

In yet another embodiment, a method includes: splitting water using acathode including a cobalt-phosphorus alloy and an anode includingcobalt phosphate in a solution containing a chemolithoautotrophicbacteria to form hydrogen (H₂) and oxygen (O₂) in the solution;providing carbon dioxide (CO₂) in the solution; and limitingbioavailable nitrogen in the solution to below a threshold to cause thechemolitoautrophic bacteria to produce a product.

In yet another embodiment, a system includes a reactor chamber with asolution contained therein. The solution may include hydrogen (H₂),carbon dioxide (CO₂), bioavailable nitrogen, and a chemolithoautotrophicbacteria. The system also includes a pair of electrodes that split watercontained within the solution to form the hydrogen. The pair ofelectrodes include a cathode including a cobalt-phosphorus alloy and ananode including cobalt phosphate. A concentration of the bioavailablenitrogen in the solution is below a threshold nitrogen concentration tocause the chemolitoautrophic bacteria to produce a product.

In yet another embodiment, a chemolithoautotrophic bacterium isresistant to reactive oxygen species.

It should be appreciated that the foregoing concepts, and additionalconcepts discussed below, may be arranged in any suitable combination,as the present disclosure is not limited in this respect. Further, otheradvantages and novel features of the present disclosure will becomeapparent from the following detailed description of various non-limitingembodiments when considered in conjunction with the accompanyingfigures.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures may be represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1A is a schematic representation of a reactor;

FIG. 1B is a schematic representation of the production of one or moreproducts within the reactor of FIG. 1A;

FIG. 2 is a table detailing experimental results for differentproduction conditions;

FIG. 3 is a graph of current voltage characteristics of different watersplitting catalysts;

FIG. 4 is a graph of current density versus potential for CoP_(i) on acarbon cloth versus CoP_(i) coated on stainless steel and a blank carboncloth;

FIG. 5 is a graph of current versus time illustrating the faradaicefficiency of CoP_(i) coated on a carbon cloth;

FIG. 6 is a graph of energy efficiencies η_(elec) and kinetics for theproduction of biomass and products at different applied potentials andsystem configurations with the solid bars indicating averages of 5-6days and dashed bars indicating 24-hr maximums;

FIG. 7 is a graph of conductivity versus salinity;

FIG. 8 is a graph of water-splitting currents in solutions of highersalinity;

FIG. 9 is a graph of optical density (OD) relative to a calibratedsignal for OD₆₀₀ indicating biomass accumulation as well as thecumulative applied electric charge that was passed versus the durationof experiments using a 100% CO₂ headspace at 1 atm and an appliedpotential of 2 V;

FIG. 10 is a graph of optical density (OD) relative to a calibratedsignal for OD₆₀₀ indicating biomass accumulation as well as thecumulative applied electric charge that was passed versus the durationof experiments while exposed to atmospheric air and an applied potentialof 2 V;

FIG. 11 is a graph of a microbial growth model that predicts linearcorrelation between electric charges and biomass accumulation;

FIG. 12 is a graph of real-time monitoring of biomass accumulation under“day”/“night” cycle testing;

FIG. 13 is a schematic reaction diagram and scanning electron microscopyimages for a Co—P alloy cathode and a CoP_(i) anode with scale bars onthe SEM images of 10 μm;

FIG. 14 is a graph of current density versus potential characteristicsof different HER catalysts (pH 7, 10 mV/sec);

FIG. 15 is a 16-day chronoamperometry graph demonstrating the stabilityof a Co—P cathode;

FIG. 16 is a graph of H₂O₂ accumulation versus time for various cathodescombined with a CoP_(i) anode with an E_(appl)=2.2 V;

FIG. 17 is a cyclic voltammetry graph of Co²⁺ and Ni²⁺ in the presenceof phosphate (P_(i)) with metal concentrations of 0.5 mM and cycled at50 mV/sec (the curve for Ni²⁺ is magnified by 50 times);

FIG. 18 is a spot assay of R. eutropha in the presence of Ni²⁺ and Co²⁺at different concentrations;

FIG. 19 is a graph of Optical Density, concentrations of PHB, andcharges passed through the electrodes plotted vs. duration at 24 hrintervals;

FIG. 20 is a graph of the averaged η_(elec) for biomass, PHB, and theoverall η_(elec) combining biomass and chemical formation at 24 hrintervals;

FIG. 21 is a graph of Optical Density, concentrations of C₃ alcohol, andcharges passed through the electrodes are plotted vs. duration at 24 hrintervals;

FIG. 22 is a graph of the averaged η_(elec) for biomass, C₃ alcohol, andthe overall η_(elec) combining biomass and chemical formation at 24 hrintervals;

FIG. 23 is a graph of Optical Density, concentrations of C₄+C₅ alcohol,and charges passed through the electrodes are plotted vs. duration at 24hr intervals;

FIG. 24 is a graph of the averaged η_(elec) for biomass, C₄+C₅ alcohol,and the overall η_(elec) combining biomass and chemical formation at 24hr intervals;

FIG. 25 is a spot assay of the tolerance of R. eutropha versus differentconcentrations of isopropanol; and

FIG. 26 is a spot assay illustrating the reactive oxygen species (ROS)tolerance between H₁₆ and ROS-resistant BC4 strains of R. eutrophabacteria.

DETAILED DESCRIPTION

The inventors have recognized that it may be desirable to operatebioreactors for either the production of materials and/or energy storagepurposes with higher efficiencies than have been achieved in the past.Additionally, the inventors have recognized that it may be desirable insome instances to enable sustained production of a desired product athigher conversion efficiencies than are achieved in typical reactors. Inview of the above, the Inventors have recognized the benefits associatedwith a reactor including H₂-oxidizing autotrophic microorganisms as wellas electrodes that split water within a solution in the reactor togenerate hydrogen or reducing equivalents within the reactor itself thatis then used by the microorganisms to perform carbon fixation to producea desired product.

In one embodiment, a system includes a reactor chamber containing asolution. The solution may include hydrogen (H₂), carbon dioxide (CO₂),bioavailable nitrogen, and a bacteria. Gasses such as one or more ofhydrogen (H₂), carbon dioxide (CO₂), nitrogen (N₂), and oxygen (O₂) mayalso be located within a headspace of the reactor chamber, thoughembodiments in which a reactor does not include a headspace such as in aflow through reactor are also contemplated. The system may also includea pair of electrodes immersed in the solution. The electrodes areconfigured to apply a voltage potential to, and pass a current through,the solution to split water contained within the solution to form atleast hydrogen (H₂) and oxygen (O₂) gasses in the solution. These gasesmay then become dissolved in the solution. During use, a concentrationof the bioavailable nitrogen in the solution may be maintained below athreshold nitrogen concentration that causes the bacteria to produce adesired product. This product may either by excreted from the bacteriaand/or stored within the bacteria as the disclosure is not so limited.

Concentrations of the above noted gases both dissolved within asolution, and/or within a headspace above the solution, may becontrolled in any number of ways including bubbling gases through thesolution, generating the dissolved gases within the solution as notedabove (e.g. electrolysis/water splitting), periodically refreshing acomposition of gases located within a headspace above the solution, orany other appropriate method of controlling the concentration ofdissolved gas within the solution. Additionally, the various methods ofcontrolling concentration may either be operated in a steady-state modewith constant operating parameters, and/or a concentration of one ormore of the dissolved gases may be monitored to enable a feedbackprocess to actively change the concentrations, generation rates, orother appropriate parameter to change the concentration of dissolvedgases to be within the desired ranges noted herein. Monitoring of thegas concentrations may be done in any appropriate manner including pHmonitoring, dissolved oxygen meters, gas chromatography, or any otherappropriate method.

As noted above, in one embodiment, the composition of a volume of gaslocated in a headspace of a reactor may include one or more of carbondioxide, oxygen, hydrogen, and nitrogen. A concentration of the carbondioxide may be between 10 volume percent (vol %) and 100 vol %. However,carbon dioxide may also be greater than equal to 0.04 vol % and/or anyother appropriate concentration. For example, carbon dioxide may bebetween or equal to 0.04 vol % and 100 vol %. A concentration of theoxygen may be between 1 vol % and 99 vol % and/or any other appropriateconcentration. A concentration of the hydrogen may be greater than orequal to 0.05 vol % and 99%. A concentration of the nitrogen may bebetween 0 vol % and 99 vol %.

As also noted, in one embodiment, a solution within a reactor chambermay include water as well as one or more of carbon dioxide, oxygen, andhydrogen dissolved within the water. A concentration of the carbondioxide in the solution may be between 0.04 vol % to saturation withinthe solution. A concentration of the oxygen in the solution may bebetween 1 vol % to saturation within the solution. A concentration ofthe hydrogen in the solution may be between 0.05 vol % to saturationwithin the solution provided that appropriate concentrations of carbondioxide and/or oxygen are also present.

As noted previously, and as described further below, production of adesired end product by bacteria located within the solution may becontrolled by limiting a concentration of bioavailable nitrogen, such asin the form of ammonia, amino acids, or any other appropriate source ofnitrogen useable by the bacteria within the solution to below athreshold nitrogen concentration. However, and without wishing to bebound by theory, the concentration threshold may be different fordifferent bacteria and/or for different concentrations of bacteria. Forexample, a solution containing enough ammonia to support a Ralstoniaeutropha population up to an optical density (OD) of 2.3 producesproduct at molar concentrations less than or equal to 0.03 M while apopulation with an OD of 0.7 produces product at molar concentrationsless than or equal to 0.9 mM. Accordingly, higher optical densities maybe correlated with producing product at higher nitrogen concentrationswhile lower optical densities may be correlated with producing productat lower nitrogen concentrations. Further, bacteria may be used toproduce product by simply placing them in solutions containing nonitrogen. In view of the above, an optical density of bacteria within asolution may be between or equal to 0.1 and 12, 0.7 and 12, or any otherappropriate concentration including concentrations both larger andsmaller than those noted above. Additionally, a concentration ofnitrogen within the solution may be between or equal to 0 and 0.2 molar,0.0001 and 0.1 molar, 0.0001 and 0.05 molar, 0.0001 and 0.03 molar, orany other appropriate composition including compositions greater andless than the ranges noted above.

While particular gasses and compositions have been detailed above, itshould be understood that the gasses located with a headspace of areactor as well as a solution within the reactor may includecompositions and/or concentrations as the disclosure is not limited inthis fashion.

Bacteria used in the systems and methods disclosed herein may beselected so that the bacteria both oxidize hydrogen as well as consumecarbon dioxide. Accordingly, in some embodiments, the bacteria mayinclude an enzyme capable of metabolizing hydrogen as an energy sourcesuch as with hydrogenase enzymes. Additionally, the bacteria may includeone or more enzymes capable of performing carbon fixation such asRibulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). One possibleclass of bacteria that may be used in the systems and methods describedherein to produce a product include, but are not limited to,chemolithoautotrophs. Additionally, appropriate chemolithoautotrophs mayinclude any one or more of Ralstonia eutropha (R. eutropha) as well asAlcaligenes paradoxs I360 bacteria, Alcaligenes paradoxs 12/X bacteria,Nocardia opaca bacteria, Nocardia autotrophica bacteria, Paracoccusdenitrificans bacteria, Pseudomonas facilis bacteria, Arthrobacterspecies 11X bacteria, Xanthobacter autotrophicus bacteria, Azospirillumlipferum bacteria, Derxia Gummosa bacteria, Rhizobium japonicumbacteria, Microcyclus aquaticus bacteria, Microcyclus ebruneus bacteria,Renobacter vacuolatum bacteria, and any other appropriate bacteria.

Depending on the particular product that it is desired to make, abacteria may either naturally include a production pathway, or may beappropriately engineered, to include a production pathway to produce anynumber of different products when placed under the appropriate growthconditions. Appropriate products include, but are not limited to: short,medium, and long chain alcohols including for example one or more ofisopropanol (C₃ alcohol), isobutanol (C₄ alcohol), 3-methyl-1-butanol(C₅ alcohol), or any other appropriate alcohol; short, medium, and longchain fatty acids; short, medium, and long chain alkanes; polymers suchas polyhydroxyalkanoates including poly(3-hydroxybutyrate) (PHB); aminoacids, and/or any other appropriate product as the disclosure is not solimited.

FIG. 1A shows a schematic of one embodiment of a system including one ormore reactor chambers. In the depicted embodiment, a single-chamberreactor 2 houses one or more pairs of electrodes including an anode 4 aand a cathode 4 b immersed in a water based solution 6. Bacteria 8 arealso included in the solution. A headspace 10 corresponding to a volumeof gas that is isolated from an exterior environment is located abovethe solution within the reactor chamber. The gas volume may correspondto any appropriate composition including, but not limited to, carbondioxide, nitrogen, hydrogen, oxygen, and any other appropriate gases asthe disclosure is not so limited. Additionally, as detailed furtherbelow, the various gases may be present in any appropriate concentrationas detailed previously. However, it should be understood thatembodiments in which a reactor chamber is exposed to an externalatmosphere that may either be a controlled composition and/or a normalatmosphere are also contemplated. The system may also include one ormore temperature regulation devices such as a water bath, temperaturecontrolled ovens, or other appropriate configurations and/or devices tomaintain a reactor chamber at any desirable temperature range forbacterial growth.

In embodiments where a reactor chamber interior is isolated from anexterior environment, the system may include one or more seals 12. Inthe depicted embodiment, the seal corresponds to a cork, stopper, athreaded cap, a latched lid, or any other appropriate structure thatseals an outlet from an interior of the reactor chamber. In thisparticular embodiment, a power source 14 is electrically connected tothe anode and cathode via two or more electrical leads 16 that passthrough one or more pass throughs in the seal to apply a potential toand pass a current I_(DC) to split water within the solution intohydrogen and oxygen through an oxygen evolution reaction (OER) at theanode and a hydrogen evolution reaction (HER) at the cathode. While theleads have been depicted as passing through the seal, it should beunderstood that embodiments in which the leads pass through a differentportion of the system, such as a wall of the reactor chamber, are alsocontemplated as the disclosure is so limited.

Depending on the particular embodiment, the above-described power sourcemay correspond to any appropriate source of electrical current that isapplied to the electrodes. However, in at least one embodiment, thepower source may correspond to a renewable source of energy such as asolar cell, wind turbine, or any other appropriate source of currentthough embodiments in which a non-renewable energy source, such as agenerator, battery, grid power, or other power source is used are alsocontemplated. In either case, a current from the power source is passedthrough the electrodes and solution to evolve hydrogen and oxygen. Thecurrent may be controlled to produce hydrogen and/or oxygen at a desiredrate of production as noted above.

In some embodiments, the electrodes may be coated with, or formed from,a water splitting catalyst to further facilitate water splitting and/orreduce the voltage applied to the solution. In some embodiments, thecatalysts may be coated onto an electrode substrate including, forexample, carbon fabrics, porous carbon foams, porous metal foams, metalfabrics, solid electrodes, and/or any other appropriate geometry ormaterial as the disclosure is not so limited. In another embodiment, theelectrodes may simply be made from a desired catalyst material. Severalappropriate materials for use as catalysts include, but are not limitedto, one or more of a cobalt-phosphorus (Co—P) alloy, cobalt phosphate(CoP_(i)), cobalt oxide, cobalt hydroxide, cobalt oxyhydroxide, a NiMoZnalloy, or any other appropriate material. As noted further below,certain catalysts offer additional benefits as well. For example, in onespecific embodiment, the electrodes may correspond to a cathodeincluding a cobalt-phosphorus alloy and an anode including cobaltphosphate, which may help to reduce the presence of reactive oxygenspecies and/or metal ions within a solution. A composition of theCoP_(i) coating and/or electrode may include phosphorous compositionsbetween or equal to 0 weight percent (wt %) and 50 wt %. Additionally,the Co—P alloy may include between 80 wt % and 99 wt % Co as well as 1wt % and 20 wt % P. However, embodiments in which different elementconcentrations are used and/or other types of catalysts and/orelectrodes are used are also contemplated as the disclosure is not solimited. For example, stainless steel, platinum, and/or other types ofelectrodes may be used.

As also shown in FIG. 1, in some embodiments, it may be desirable toeither continuously, or periodically, bubble, i.e. sparge or flush, oneor more gases through a solution 6 and/or to refresh a composition ofgases located within a head space 10 of the reactor chamber 2 above asurface of the solution. In such an embodiment, a gas source 18 may bein fluid communication with one or more gas inlets 20 that pass througheither a seal 12 and/or another portion of the reactor chamber 2 such asa side wall to place the gas source in fluid communication with aninterior of the reactor chamber. Additionally, in some embodiments, oneor more inlets discharge a flow of gas into the solution so that the gaswill bubble through the solution. However, embodiments in which the oneor more gas inlets discharge a flow of gas into the headspace of thereactor chamber instead are also contemplated as the disclosure is notso limited. Additionally, one or more corresponding gas outlets 22 maybe formed in a seal and/or another portion of the reactor chamber topermit a flow of gas to flow from an interior to an exterior of thereactor chamber. It should be noted that gas inlets and outlets maycorrespond to any appropriate structure including, but not limited to,tubes, pipes, flow passages, ports in direct fluid communication withthe reactor chamber interior, or any other appropriate structure.

Gas sources may correspond to any appropriate gas source capable ofproviding a pressurized flow of gas to the chamber through the inletincluding, for example, one or more pressurized gas cylinders. While agas source may include any appropriate composition of one or moregasses, in one embodiment, a gas source may provide one or more ofhydrogen, nitrogen, carbon dioxide, and oxygen. The flow of gas providedby the gas source may have a composition equivalent to the range of gascompositions described above for the gas composition with a headspace ofthe reactor chamber. Further, in some embodiments, the gas source maysimply be a source of carbon dioxide. Of course embodiments in which adifferent mix of gases, other including different gases and/or differentconcentrations than those noted above, is bubbled through a solution orotherwise input into a reactor chamber are also contemplated as thedisclosure is not so limited. Additionally, the gas source may be usedto help maintain operation of a reactor at, below, and/or aboveatmospheric pressure as the disclosure is not limited to any particularpressure range.

The above noted one or more gas inlets and outlets may also include oneor more valves located along a flow path between the gas source and anexterior end of the one or more outlets. These valves may include forexample, manually operated valves, pneumatically or hydraulicallyactuated valves, unidirectional valves (i.e. check valves) may also beincorporated in the one or more inlets and/or outlets to selectivelyprevent the flow of gases into or out of the reactor either entirely orin the upstream direction into the chamber and/or towards the gassource.

While the use of inlet and/or outlet gas passages have been describedabove, embodiments in which there are no inlet and/or outlets for gassesare present are also contemplated. For example, in one embodiment, asystem including a sealable reactor may simply be flushed withappropriate gasses prior to being sealed. The system may then be flushedwith an appropriate composition of gasses at periodic intervals torefresh the desired gas composition in the solution and/or headspaceprior to resealing the reactor chamber. Alternatively, the head spacemay be sized to contain a gas volume sufficient for use during an entireproduction run.

In instances where electrodes are run at high enough rates and/or forsufficient durations, concentration may be formed within a solution in areactor chamber. Accordingly, it may be desirable to either preventand/or mitigate the presence of concentration gradients in the solution.Therefore, in some embodiments, a system may include a mixer such as astir bar 24 illustrated in FIG. 1A. Alternatively, a shaker table,and/or any other way of inducing motion in the solution to reduce thepresence of concentration gradients may also be used as the disclosureis not so limited.

While the above embodiment has been directed to an isolated reactorchamber, embodiments in which a flow-through reaction chamber with twoor more corresponding electrodes immersed in a solution that is flowedthrough the reaction chamber and past the electrodes are alsocontemplated. For example, one possible embodiment, one or morecorresponding electrodes may be suspended within a solution flowingthrough a chamber, tube, passage, or other structure. Similar to theabove embodiment, the electrodes are electrically coupled with acorresponding power source to perform water splitting as the solutionflows past the electrodes. Such a system may either be a single passflow through system and/or the solution may be continuously flowedpassed the electrodes in a continuous loop though other configurationsare also contemplated as well.

Without wishing to be bound by theory, FIG. 1B illustrates one possiblepathway for a system to produce one or more desired products. In thedepicted embodiment, the hydrogen evolution reaction occurs at thecathode 4 b. During the reaction at the cathode, two hydrogen ions (H⁺)are combined with two electrons to form hydrogen gas H₂ that dissolveswithin the solution 6 along with carbon dioxide (CO₂), which dissolvedin the solution as well. At the same time various toxicants such asreactive oxygen species (ROS) including, for example, hydrogen peroxide(H₂O₂), superoxides (O₂.⁻), and/or hydroxyl radical (HO.) species aswell as metallic ions may be generated at the cathode. For example, Co²⁺ions may be dissolved into solution when a cobalt based cathode is used.As described further below, in some embodiments, the use of certaincatalysts may help to reduce the production of ROS and the metallic ionsleached into the solution may be deposited onto the anode using one ormore elements located within the solution to form compounds such as acobalt phosphate.

As also illustrated in FIG. 1B, once hydrogen and carbon dioxide areprovided within a solution, bacteria 8 present within the solution maybe used to transform these compounds into useful products. For example,in one embodiment, the bacteria uses hydrogenase to metabolize thedissolved hydrogen gas and one or more appropriate enzymes, such asRuBisCO or other appropriate enzyme, to provide a carbon fixationpathway. This may include absorbing the carbon dioxide and formingAcetyl-CoA through the Calvin cycle as shown in the figure. Further,depending on the concentration of nitrogen within the solution, thebacteria may either form biomass or one or more desired products. Forinstance, if a concentration of nitrogen within the solution is below apredetermined nitrogen concentration threshold, the bacteria may formone or more products such as the C3, C4, and/or C5 alcohols, PHB, and/orcombinations of the above depicted in the figure.

Depending on the embodiment, a solution placed in the chamber of areactor may include water with one or more additional solvents,compounds, and/or additives. For example, the solution may include:inorganic salts such as phosphates including sodium phosphates andpotassium phosphates; trace metal supplements such as iron, nickel,manganese, zinc, copper, and molybdenum; or any other appropriatecomponent in addition to the dissolved gasses noted above. In one suchembodiment, a phosphate may have a concentration between 9 and 90 mM, 9and 72 mM, 9 and 50 mM, or any other appropriate concentration. In aparticular embodiment, a water based solution may include one or more ofthe following in the listed concentrations: 12 mM to 123 mM of Na₂HPO₄,11 mM to 33 mM of KH₂PO₄, 1.25 mM to 15 mM of (NH₄)₂SO₄, 0.16 mM to 0.64mM of MgSO₄, 2.4 μM to 5.8 μM of CaSO₄, 1 μM to 4 μM of NiSO₄, 0.81 μMto 3.25 μM molar concentration of Ferric Citrate, 60 mM to 240 mM molarconcentration of NaHCO₃.

As noted above in regards to the discussion of FIG. 1B, reactive oxygenspecies (ROS) as well as metallic ions may be formed and/or dissolvedinto a solution during the hydrogen evolution reaction at the cathode.However, ROS and larger concentrations of the metallic ions within thesolution may be detrimental to cell growth above certain concentrations.It is noted that the use of continuous hydrogen production within areactor to form hydrogen for conversion into one or more desiredproducts has been hampered by the production of these ROS and metallicion concentrations because the bacteria used to form the desiredproducts tend to be sensitive to these compounds and ions limiting thegrowth of, and above certain concentrations, killing the bacteria.Therefore, in some embodiments, it may be desirable to apply voltages,use electrodes that produce less ROS, remove and/or prevent thedissolution of metallic ions from the electrodes, and/or use bacteriathat are resistant to the presence of these toxicants as detailedfurther below.

As noted above, it may be desirable to select one or more catalysts foruse as the electrodes that produce fewer reactive oxygen species (ROS)during use. Specifically, a biocompatible catalyst system that is nottoxic to the bacterium and lowers the overpotential for water splittingmay be used in some embodiments. One such example of a catalyst includesa ROS-resistant cobalt-phosphorus (Co—P) alloy cathode. This cathode maybe combined with a cobalt phosphate (CoP_(i)) anode. This catalyst pairhas the added benefit of the anode being self-healing. In other words,the catalyst pair helps to remove metallic Co²⁺ ions present with asolution in a reactor. Without wishing to be bound by theory, theelectrode pair works in concert to remove extracted metal ions from thecathode by depositing them onto the anode which may help to maintainextraneous cobalt ions at relatively low concentrations within solutionand to deliver a low applied electrical potential to split water togenerate H₂. Without wishing to be bound by theory, it is believed thatduring electrolysis of the water, phosphorus and/or cobalt is extractedfrom the electrodes. The reduction potential of leached cobalt is suchthat formation of cobalt phosphate using phosphate available in thesolution is energetically favored. Cobalt phosphate formed in solutionthen deposits onto the anode at a rate linearly proportional to freeCo²⁺, providing a self-healing process for the electrodes. In view ofthe above, the cobalt-phosphorus (Co—P) alloy and cobalt phosphate(CoP_(i)) catalysts may be used to help mitigate the presence of bothROS and metal ions within the solution to help promote growth ofbacteria within the reactor chamber.

It should be understood that any appropriate voltage may be applied to apair of electrodes immersed in a solution to split water into hydrogenand oxygen. However, in some embodiments, the applied voltage may belimited to fall between upper and lower voltage thresholds. For example,the self-healing properties of a cobalt phosphate and cobalt phosphorousbased alloy electrode pair may function at voltage potentials greaterthan about 1.42 V. Additionally, the thermodynamic minimum potential forsplitting water is about 1.23 V. Therefore, depending on the particularembodiment, the voltage applied to the electrodes may be greater than orequal to about 1.23 V, 1.42 V, 1.5 V, 2 V, 2.2 V, 2.4 V, or any otherappropriate voltage. Additionally, the applied voltage may be less thanor equal to about 10 V, 5 V, 4 V, 3 V, 2.9 V, 2.8 V, 2.7 V, 2.6 V, 2.5V, or any other appropriate voltage. Combinations of the above notedvoltage ranges are contemplated including, for example, a voltageapplied to a pair of electrodes may be between 1.23 V and 10 V, 1.42 Vand 5 V, 2 V and 3 V, 2.3 V and 2.7 V as well as other appropriateranges. Additionally, it should be understood that voltages both greaterthan and less than those noted above, as well as different combinationsof the above ranges, are also contemplated as the disclosure is not solimited. In addition to the applied voltages, any appropriate currentmay be passed through the electrodes to perform water splitting whichwill depend on the desired rate of hydrogen generation for a givenvolume of a reactor being used. For example, in some embodiments, acurrent used to split water may be controlled to generate hydrogen at arate substantially equal to a rate of hydrogen consumption by bacteriain the solution. However, embodiments in which hydrogen is produced atrates both greater than or less than consumption by the bacteria arealso contemplated.

In addition to using catalysts, controlling the solution pH, andapplying appropriate driving potentials, and/or controlling any otherappropriate parameter to reduce the presence of reactive oxygen species(ROS) within the solution in a reaction chamber, it may also bedesirable to use bacteria that are resistant to the presence of ROSand/or metallic ions present within the solution as noted previously.Specifically, a chemolithautotrophic bacterium that is resistant toreactive oxygen species may be used. Further, in some embodiments a R.eutropha bacteria that is resistant to ROS as compared to a wild-typeH16 R. eutropha may be used. Table I below details several geneticpolymorphisms found between the wild-type H16 R. eutropha and aROS-tolerant BC4 strain that was purposefully evolved during theexperiments detailed below. Mutations of the BC4 strain relative to thewild type bacteria are detailed further below.

Two single nucleotide polymorphisms and two deletion events wereobserved. Without wishing to be bound by theory, the large deletion fromaciC1 may indicate a decrease in overall membrane permeability, possiblyaffecting superoxide entry to the cell resulting in the observed ROSresistance. The genome sequences are accessible at the NCBI SRA databaseunder the accession number SRP073266 and specific mutations of the BC4strain are listed below in Table 1. The standard genome sequence for thewild-type H16 R. eutropha is also accessible at the RCSB Protein DataBank under accession number AM260479 which the following mutations mayalso be referenced to.

TABLE I Mutation Position Annotation Gene Description G → T 611,894R133R acrC1 cation/multidrug efflux system outer membrane protein Δ45 bp611,905 344-388 of 1494 acrC1 cation/multidrug efflux nt system outermembrane protein G → A 2,563,281 intergenic, Hfq and uncharacterizedhost factor (−1/+210) H16_A2360 I protein/GTP-binding protein Δ15 bp241,880 363-377 of 957 H16_B021 transcriptional regulator, nt 4LysR-Family

In reference to the above table, an R. eutropha bacteria may include atleast one to four mutations selected from the mutations noted above inTable 1 and may be selected in any combination. These specific mutationsare listed below in more detail with mutations noted relative to thewild type R. eutropha bolded and underlined within the sequences givenbelow.

The first noted mutation may correspond to the sequence listed belowranging from position 611790-611998 for Ralstonia eutropha H16chromosome 1.

(SEQ ID NO: 1) GCCTCGCTGCTTTCCACCTGGCGCCGCACGCGGCCCCAGACGTCGATTTCCCAGGTTGCGCCCAGGGTCGCGCTCTGCCCGTTGAGCGTGCTGCCG CTGGCGCC GCGCGCGCGCGAGGCGCCGGCCTGTGCGTCGACGGTCGGGAAGAAGCCGGCGCGCGCGGCCTGCAGCGACGCCACCGCCTGGCGGTACTGCG CCTCGGCGGCCTT

The second noted mutation may correspond to the sequence listed belowranging from position 611905-613399 for Ralstonia eutropha H16chromosome 1.

(SEQ ID NO: 2) AGGCGCCGGCCTGTGCGTCGACGGTCGGGAAGAAGCCGGCGCGCGCGGCCTGCAGCGACGCCACCGCCTGGCGGTACTGCGCCTCGGCGGCCTTGATGTTCTGGTTCGAGATCTGCACCTCGGACATCAGCGCGTCGAGCTGCGCATCGCCGAACACGGTCCACCAGTCGGCGCGTGCCAGCGCATCCTGCGGCTCGGCGGGCTTCCAGTCGCCGGTCCAGGCGGGGGTGGCGGCATCGGCTTCCTTGAAGGATGCGGAAACCGGCGCGTCGGGGCGCTGGTAGTCGGGGCCGACGGCGCAGCCGGCCAGCAGCAGCGCGCAGGCCAGCGACACCGGCAGGGCA TGGGTCAGGAGGCGGGAAAGAACTGTCATGTCGAGTCTTCGCAAAT CTAGACGGCGGCCGGCTGGTCAGGCGTGCCGGCACCACGGCGGCGCTGGCGCCAGGCCTTGACCTTCAGGCGCCAGCGGTCCAGCGTCAGGTAGACCACCGGCGTGGTGTACAGCGTCAGCAGCTGGCTTACCACCAGTCCGCCGACAATGGAGATGCCCAGCGGCGCGCGCAGTTCGGCGCCGTCGCCGCGGCCGATTGCCAGCGGCACCGCGCCCAGCAGCGCGGCCATGGTGGTCATCAGGATCGGGCGGAAGCGCAGCAGGCAGGCGCGGTAGATCGCGTCGCGCGGCGACAGGCCATCGCGCCGTTCGGCATCGATGGCGAAGTCGATCATCATGATCGCGTTCTTTTTCACGATGCCGATCAGCAGGATCACGCCGATCAGCGCGATGATGCTGAAGTCGGTCTTCGATGCCAGCAGCGCCAGCAGCGCGCCCACGCCGGCGGAGGGCAGCGTCGACAGGATCGTCAGCGGATGCACATAGCTTTCATACAGCACGCCCAGCACGATGTAGATCGTGATCAGCGCCGCCAGGATCAGGATCGGCTGACTCTTGAGCGAATCCTGGAACGCCTTGGCGCCGCCCTGGAAGTTGGCGCGCAGCGTCTCCGGCACGCCGATGCGCGCCATCTCGCGCGTGATCGCGTCGGTCGCCTGCGACAGCGAAGTGCCCTCGGCCAGGTTGAACGAGATCGTCGAGGCCGCGAACTGGCCCTGGTGGTTCACGCCCAGCGGCGTGCTGGACGGGGTCACGCGCGCGAACGCCGCCAGCGGCACGCGGTTGCCGTTGCCGGTGACCACGTAGATGTCCTTGAGCGCATCGGGCCCTTGCAGGTATTCCTGGCTCAGCTCCATCACCACGCGGTACTGGTTCAGCGGATGGTAGATGGTGGACACCAGCCGCTGGCCGAAGGCATCGTTGAGCACCGCATCCACCTGCTGCGCGGTCACGCCCAGGCGCGAGGCCGCGTCGCGGTCGATGATCACCGAGGTCTGCAGGCCCTTGTCGTTGGTATCGGTGTCGATATCCTCCAGCCCCTTCAGGTTCGACAACGCGGCGCGCACCTTGGGCTCCCACGCGCGCAGCACTTCCAGGTCGTCC

The third noted mutation may correspond to the sequence listed belowranging from position 2563181-2563281 for Ralstonia eutropha H16chromosome 1.

(SEQ ID NO: 3) GCAGCTTGATGCCATTGACGAGGTAGATGGAAACCGGCACGTGCTCTTTGCGCAGCGCGTTCAGGAACGGGCCTTGTAGCAGTTGCCCTTTGTTG CTCAT GGCACACTCCAAATTTATAGGTTTAGTGGTGAATGATGGGGATGGAAATCCCCGGTTCAAGTCAGGCGGCGCAAAAACGCGCCAGAAAAAAGA TCAAAAAC

The fourth noted mutation may correspond to the sequence listed belowranging from position 241880-242243 for Ralstonia eutropha H16chromosome 1.

(SEQ ID NO: 4) GAGGATGCCATGTCCGAAGCGCCTGTCCTTGCCCCCTCGACCTCAACCCAGCCGCCCGCCGCCGGCCAGCTCAACCTGATCCGCCCGCAGCCATATGCCGACTGGGCGCCGCAGGTCACGGCCGAAGAACGCGCCACGCTGCGCCGCGAGCTGGAGCAGGGCGCCGTGCTGTACTTCCCGAACCTGAATTTCCGCTTCCAGCCGGGCGAAGAGCGCTTCCTTGACAGCCGCTATTCCGACGGCAAGTCCAAGAACATCAACCTGCGCGCCGACGACACCGCGGTGCGCGGCGCCCAGGGCAGTCCGCAGGACCTGGCGGACCTGTACACGCTGATCCGCCGCTACG CCGACAACAGCGAATTGCTGGTGCGCACGCTGT TCCCTGAATACATCCCGCACATGACGCGCGCCGGCACCTCGCTGCGGCCCAGCGAGATCGCCGGGCGCCCGGTCAGCTGGCGCAAGGACGACACCCGCCT

In the above sequences, it should be understood that a bacteria mayinclude changes in one or more base pairs relative to the mutationsequences noted above that still produce the same functionality and/oramino acid within the bacteria. For example, a bacteria may include 95%,96%, 97%, 98%, 99%, or any other appropriate percentage of the samemutation sequences listed above while still providing the noted enhancedROS resistance.

As elaborated on in the examples, the systems described herein arecapable of undergoing intermittent production. For example, when adriving potential is applied to the electrodes to generate hydrogen, thebacteria produce the desired product. Correspondingly, when thepotential is removed and hydrogen is no longer generated, production ofthe product is ceased once the available hydrogen is consumed and areduction in overall biomass is observed until the potential is onceagain applied to the electrodes to generate hydrogen. The system willthen resume biomass and/or product formation. Thus, while a system maybe run continuously to produce a desired product, in some modes ofoperation a driving potential may be intermittently applied to theelectrodes to intermittently split water to form hydrogen andcorrespondingly intermittently produce a desired product. A frequency ofthe intermittently applied potential may be any frequency and may eitherbe uniform or non-uniform as the disclosure is not so limited. Thisability to intermittently produce a product may be desirable inapplications such as when intermittent renewable energy sources are usedto provide the power applied to the electrodes including, but notlimited to, intermittent power sources such as solar and wind energy.

EXAMPLES: EXPERIMENTAL SYSTEMS

The solution used during the experiments described herein was preparedaccording to the following procedure. A first solution was preparedusing 940 mL deionized H2O, 6.74 g Na₂HPO₄-7H₂O, 1.5 g KH₂PO₄, and 1.0 g(NH₄)₂SO₄. A second solution was prepared with 400 mL deionized H₂O, 4.0g MgSO₄-7H₂O, 50 mg CaSO₄-2H₂O (stirred extensively to dissolve), and 28mg NiSO₄-7H₂O. A third solution was prepared with 400 mL deionized H₂Oand 20 mg Ferric citrate. A fourth solution was prepared using 400 mLdeionized H₂O and 10.0 g NaHCO₃. Without wishing to be bound by theory,the first, second, and fourth solutions were filtered and sterilized.The third solution was not. The second, third, and fourth solutionswhere then combined with the first solution and mixed to combine.

The above described media had an overall phosphate salt concentration of36 mM which may be decreased to about 9 mM before the weakly bufferedsolution began to deteriorate the carbon cloth anode during use.However, different concentrations may be useable with different anodesubstrate materials. Additionally, it was found that R. eutropha wascapable of tolerating 72 mM phosphate salt (2×) but died at a phosphatesalt concentrations greater than or equal to 108 mM (3×) reliably.Though again, different appropriate solution concentrations may be usedfor different bacteria and/or when used with different solutioncompositions. At 36 mM phosphate salts, the pH of the solution withunder 400 ppm CO₂ was 7. R. eutropha media typically range from pH=6-8but hydrogenase has been shown to operate as low as pH=4.5. At a 100%CO₂ headspace at 1 atm, the final pH was about 6.2. Without wishing tobe bound by theory, the sodium bicarbonate helped to maintain osmoticpressure and ionic strength. Therefore, a concentration of the sodiumbicarbonate was balanced based on the pH.

The cobalt phosphorous alloy and cobalt phosphate catalysts for hydrogenevolution reaction (HER) and oxygen evolution reaction (OER) werecreated by electrochemical deposition methods with the use of a GamryInterface 1000 potentiostat. A classic three-electrode setup was appliedwith a Ag/AgCl, 1 M KCl reference electrode. After depositing thecatalysts, the electrodes were rinsed with ample deionized water.

Water splitting and bacterial CO₂ fixation took place in a singleenclosed chamber filled with CO₂ in the headspace similar to the chambershown in FIG. 1 above. The reported data are based on at least threebiological replicates (n≥3). A Gamry Reference 600 potentiostat coupledwith an ECM8 electrochemical multiplexer allowed for parallelexperiments with 8 individual reactors. The reactors included a 250 mLDuran® GL 45 glass bottle capped with a Duran® GL 45 3-ports (GL 14)connection system. The glass bottles were immersed in a 30° C. waterbath. Two of the GL 14 screw cap ports on each reactor served as thefeedthroughs for the two water-splitting electrodes, and a third feedthrough was used as a gas inlet regulated by a quarter-turn valve. For atypical experiment, 100 mL of all-inorganic minimal media solution wasadded into the reactor and water splitting was performed via atwo-electrode system; the electrodes had a 4 cm² geometric area. Theapplied potential, E_(appl), was defined as the voltage differencebetween the working and counter/reference electrodes in a two-electrodeconfiguration; E_(appl) is detailed in the table shown in FIG. 2 foreach experiment.

After inoculation with R. eutropha strains (initial OD₆₀₀=0.2), thereactor was purged with CO₂ and then sealed. The experiments werestirred at 350 rpm by a triangular stirring rod to facilitate masstransport within the reactors. The bioelectrochemical reactor headspacewas sampled daily using Supel™ inert foil gas sampling bags. Theelectrolyte was also sampled daily to quantify OD₆₀₀ and product titers.After sampling, the reactor headspace was sparged to refill CO₂ in theheadspace. In the case of CO₂ reduction directly from air, the reactorchamber was not isolated to the environment. The gas inlet port wasconnected to the ambient atmosphere through a 0.2 μm PVDF gas filter andno CO₂ gas flow was supplied to the reactor headspace. In this setup,the reactor headspace was in direct exchange with ambient environmentthrough the PVDF filter.

Of course, while isolated batch reactor designs were used in theexperiments, the batch reactor design may be modified to a flow-basedconfiguration, in which microbe-containing media would be forced to flowthrough a chamber where water splitting occurs. As detailed furtherbelow, the low level of residual H₂ gas measured in the headspaceindicates the efficient uptake of H₂ and accordingly a small energy losswould be expected under a flow reactor configuration making it a viabledesign choice.

Real-time monitoring of biomass accumulation was accomplished bymeasuring the optical density of the 100 mL reactors on a home-builtsetup at 20 sec time intervals. Specifically, a 650 nm laser pointer(Digi-Key Electronic) was directed at a photodiode across the 100 mLreactor containing bacteria and water splitting electrodes. Controlledthrough a customized script in MATLAB, every 20 sec the intensity ofincident light after scattering the culture was determined with the helpof an operational amplifier (Digi-Key Electronic). A standard curvebetween the measured light intensity and OD₆₀₀ was established, aftermeasuring the transmitted light from R. eutropha cultures of known OD₆₀₀values.

In some experiments, a 10-fold scale-up experiment was conducted. Inthese experiments, a similar reactor was used but included a 1000 mLvolume reactor. Other than as noted below, the procedure was similar tothat employed for experiments using the 100 mL reactor. A Vacu-Quik jarsystem (Almore International, inc.) was modified with two currentfeedthroughs for water splitting electrodes and two PEEK tubes as gasinlet/outlet. A Neslab EX-211 constant temperature bath circulated wateraround the jar to maintain a temperature of 30° C. For optimizedtemperature homogeneity, the jar and the water circulation was embeddedin a thermally insulating layer. The volume of minimal medium solutionwas 1000 mL, and the size of the electrodes was increasedproportionally. During the experiments, the reactor was inoculated withR. eutropha (OD₆₀₀=0.05) and grown under H₂/CO₂/air overnight. BeforeCO₂ reduction was begun, the initial OD₆₀₀ referenced signal of thebacterial optical density was recorded. The measured signal wastypically between 0.15 and 0.20 times the reference OD₆₀₀ signal. Theheadspace of the reactor was then thoroughly purged with CO₂ to removethe residual H₂ that remained from the autotrophic growth.

Facilitating CO₂ mass transport by pressurization was not observed to bebeneficial in the current experiments. However, under some conditions,such as during the use of larger reactors, pressurized reactor operationmay be beneficial.

Example: Bacterial Strains and Growth Protocols

Unless noted otherwise, the composition of the minimal medium used was6.74 g/L Na₂HPO₄.7H₂O, 1.5 g/L KH₂PO₄, 1.0 g/L (NH₄)₂SO₄, 80 mg/LMgSO₄.7H₂O, 1 mg/L CaSO₄.2H₂O, 0.56 mg/L NiSO₄.7H₂O, 0.4 mg/L ferriccitrate, and 200 mg/L NaHCO₃. Because nitrogen (N), phosphorous (P) andsulfur (S) are contributing about 10% (for N) and less than 5% (for Pand S) of the total dry cell weight, the requirement of inorganicelements is not limiting the CO₂-reduction process under the listedexperimental conditions. The constant renewal of “living” biocatalystsdoes not factor into the consumption of inorganic elements, because newbacteria can recycle the elements released from the expired microbes.This medium composition had a phosphate buffer concentration of 36 mM.To induce nitrogen-limited growth of the bacteria to produce isopropanoland PHB, the (NH₄)₂SO₄ concentration was reduced to 0.167 g/L. Forexperiments with higher salinity, the buffer strength of phosphate wasincreased by three times: 20.22 g/L Na₂HPO₄.7H₂O, 4.5 g/L KH₂PO₄. This“high salt” medium had a phosphate buffer concentration of 108 mM. Allsolutions were filter-sterilized prior to use except the ferric citratecomponent, which was added after the filter sterilization step.

NaHCO₃ was also added to the initial media preparation to maintain ionicstrength and osmotic pressure. As a conjugate base under equilibriumwith CO₂ in aqueous solution, bicarbonate can serve as a carbon sourcefor CO₂ reduction. R. eutropha converts bicarbonate to CO₂ throughcarbonic anhydrase. The prepared media was fully equilibrated before anyexperiments were conducted.

R. eutropha H16 (wild type), Re2133-pEG12, and Re2410-pJL26 strains wereobtained from Sinskey laboratory at MIT. Additionally, as detailedbelow, a ROS-resistant strain (BC4) was isolated that was evolved after11 consecutive days of exposure to a stainless steel cobalt phosphatewater-splitting system with an applied electrode potential of 2.3 V.During the development of this ROS resistant strain, no growth wasobserved until day 7 when the OD₆₀₀ rose from 0.15 to 1.15 over the next4 days. Isolated strains were sequenced and mutations were compiledusing breseq.

Unless noted otherwise, all of the microbial growth was conducted at 30°C. In general, individual colonies were picked from agar plates andinoculated into rich broth media solutions for overnight growth.Cultures were centrifuged and re-suspended in minimal mediumsupplemented with gentamicin (10 μg/mL). The cultures were placed in aVacu-Quick jar filled with H₂ (8 mmHg) and CO₂ (2 mmHg) with air asbalance. At this condition, R. eutropha adapted to autotrophicmetabolism with H₂.

Example: Toxicant Quantification

Abiotic water splitting was performed in the bioelectrochemical reactorsdescribed above using minimal medium solution as the electrolyte with anumber of electrode combinations. 50 μL of electrolyte was thentransferred at a series of time points to a 96-well plate (Corning). Theplate was kept on ice, in the dark, for no more than 1 hour prior tomeasurement. The H₂O₂ concentration was assayed using an Amplex Red H₂O₂detection kit (Sigma-Aldrich) by monitoring the absorbance at 555 nmusing a BIO-13 Synergy H1m plate reader. The concentration of H₂O₂ wasquantified by comparing against a standard curve generated from H₂O₂standards ranging from 0 to 40 μM.

The leaching rates of various elements from the electrodes was alsomeasured with inductively coupled plasma mass spectrometry (ThermoElectron, X-Series ICP-MS with collision cell technology, CCT). Afterrunning the abiotic water-splitting experiments for 24 hours at constantE_(appl), 0.5 mL of electrolyte was sampled and diluted with 3.5 mL of2% double distilled nitric acid (Sigma-Aldrich). Samples along withcalibration standards were scanned twice for 60 sec each for ⁶⁰Ni, ⁵⁹Co,and ¹⁹⁴Pt. To demonstrate the “self-healing” effect of CoP_(i) anode onthe metals leached from cathode, experiments were conducted in both one-and two-compartment electrochemical cells. In the single-compartmentsetup, both the HER cathode and OER anode were immersed in the samereactor. In the two-compartment setup (H-cell), a glass frit junction offine porosity separated the two chambers and hindered the mass transportof leached metal ions.

The procedure for spot assays was performed by diluting 100 μL ofculture grown under different conditions by 1:10 in fresh minimalmedium, which was vortexed. Three to four serial 10-fold dilutions weremade and 2 μL of each dilution was spotted on rich broth agar plates andallowed to dry. Plates were typically grown for 2 days at 30° C. beforeimaging. The half maximal inhibitory concentration (IC₅₀) was estimatedbased on the comparison at 1/100 dilution. The areas of colonies atcertain conditions were then compared with that of control samples.

Example: CO₂ Fixation

Using the biocompatible Co—P|CoP_(i) water splitting catalysts notedabove along with R. eutropha resulted in a system capable of performingCO₂-fixation using continuous H₂ production via water splitting. Forthese experiments, the CoP_(i) catalyst was deposited on ahigh-surface-area carbon cloth as the electrode support. Thisconfiguration resulted in relatively high currents as compared tostainless steel paired with a CoP_(i) electrode and a platinum electrodepair, FIG. 3. As shown in FIGS. 4 and 5, a faradaic efficiency of theelectrodes was 96±4%. During use, CO₂ reduction proceeded under aconstant voltage within the batch reactor similar to the one describedabove relative to FIG. 1. The batch reactor was half-filled with asolution containing only inorganic salts (mostly phosphate) and tracemetal supplements.

The table shown in FIG. 2 illustrates the results from multipleexperiments including different electrodes, bacterium strains, appliedvoltages, volumes, and solution compositions. Efficiencies and titersfor different E_(appl) and other experimental conditions were averagedover 5-6 days unless specifically noted otherwise in the table. As shownin the table and FIG. 6, using a CoP_(i)|Co-P electrode pair incombination with R. eutropha, the system was able to store over half itsinput energy as products of CO₂-fixation at low E_(appl). Entries 1-3and 5 show that η_(elec) increases with decreasing E_(appl) under 100%CO₂ until E_(appl)<2.0 V. Below E_(appl)=2.0 V (Entry 8). A higher saltconcentration (108 mM phosphate buffer) was also tried to facilitatemass transport and attendant current, FIGS. 7 and 8. However, highersalt concentrations were found to limit R. eutropha metabolism. Thus aconcentration of 36 mM phosphate and E_(appl)=2.0 V resulted in anoptimal η_(elec) for these experiments, though different concentrationswith different bacteria, solutions, and/or system configurations mayalso occur. The highest observed η_(elec) achieved for biomassproduction in these experiments was 54±4% (Entry 5, n=4) over a durationof 6 days.

The measured CO₂ reduction efficiency observed in the currentexperiments was comparable to the highest demonstrated by R. eutrophaduring H₂-fermentation. This biomass yield is equivalent to assimilatingabout 4.1 mol (180 g) of CO₂ captured at the cost of 1 kWh ofelectricity. The amount of captured CO₂ is 1/10 of that caught byamine-based carbon capture and storage (˜2000 g at the cost of 1 kWh),but whose processed product cannot be used as a fuel. As shown by thetable in FIG. 2, enlarging the batch reactor volume by 10-fold did notperturb the efficiency (Entries 4 and 6), indicating that the system isscalable and the reactor volume does not pose immediate limits.Interestingly, the η_(elec) under air (400 ppm CO₂) is 20±3% (Entry 7,n=3), which is only 2.7 times lower than the case of pure CO₂ in thehead space although the partial pressure of CO₂ is reduced by 2,500times. Without wishing to be bound by theory, this indicates that CO₂ isnot a limiting reagent. The roughly 20% η_(elec) for biomass conversionis equivalent to assimilating about 1.5 mol of CO₂ captured from about85,200 liters of air at ambient condition with the cost of 1 kWh ofelectricity.

The current experiments also confirmed that biomass accumulation scaleslinearly with the amount of charge passed under a pure CO₂ head space,FIG. 9 as well as when a batch reactor is exposed to the CO₂ levelsfound in ambient air FIG. 10. Without wishing to be bound by theory, thelinear growth is accounted for by a model that combines governingequations for H₂-generation from water splitting and biomassaccumulation from carbon-fixation. The model predicts a linearcorrelation between biomass and charge passed after an induction periodof low population density of bacteria and high H₂ concentration, FIG.11, which is consistent with the data shown in FIGS. 9 and 10 where theinduction period is too short to be observed. Gas chromatographymeasurements revealed a H₂ concentration in the reactor headspace of0.19±0.04% (n=3) for 100% CO₂ and 0.10±0.05% (n=3) in air, whichcorresponded to 1.5±0.3 and 0.8±0.4 μM in water. These concentrations ofH₂ are well below the Michaelis constant of about 6 μM formembrane-bound hydrogenases in R. eutropha. This may suggest that H₂ isfacilely consumed by R. eutropha. Moreover, similar linear growthconditions for both pure and ambient CO₂ atmospheres may indicate thatH₂ oxidation rather than CO₂ reduction is rate-limiting forbiosynthesis. Additionally, direct CO₂ reduction from air highlights therelatively high affinity of R. eutropha for CO₂ at low pressures and athigh O₂ concentrations, in contrast to the previously reported resultsof synthetic catalysts, individual enzymes, and strictly anaerobicorganisms such as acetogens and methanogens.

In addition to the above, as shown in FIG. 12, R. eutropha halted growthduring “night” cycles, when power was not applied to the electrodessimilar to what may occur when a system is coupled with a solar powersource, and continued CO₂ reduction 12 hr later upon resumption of thewater-splitting reaction during a corresponding day cycle when power wasapplied to the electrodes. Specifically, as shown in the figure, theOD₆₀₀ signal decreased during the “dark” phase of the day/night cycleexperiments was due to a loss of biomass. Without a source of energyduring lithotrophic growth, cells lyse and OD₆₀₀ drops. The observedgrowth, i.e. increase in biomass, in the first “night” phase may be dueto the residual, unconsumed H₂ that remains from the previous “day”phase. With higher culture density, there was no excessive H₂ in thesecond and third “night” phases, and OD₆₀₀ subsequently decreased. Thedifferences in biomass loss between the second and third “night” phasesis likely due to the conditioning of H₂ scarcity as the populationgrows. These observations confirm the intrinsic dependence of R.eutropha on H₂ generation. These data also reveal that theCoP_(i)|Co-P|R. eutropha hybrid system are compatible with theintermittent nature of a solar, or other intermittent renewable, energysource.

Example: Catalyst Function

As noted previously, a catalyst including a cobalt phosphorous alloy(Co—P) and a cobalt phosphate (CoP_(i)) may be used. As illustrated inFIG. 13, the Co—P HER and CoP_(i) OER catalysts work in synergy to forma biocompatible water splitting system that salvages Co²⁺ cationsleached from the electrodes, see FIG. 13, via pathway 2 while helping toreduce the production of various reactive oxygen species including H₂O₂along pathway 1.

Testing was conducted on the Co—P alloy cathode, which is known topromote HER under alkaline solutions, and exhibits high HER activity inwater at neutral pH with minimal ROS production. X-ray photoelectronspectroscopy of Co—P thin films supports the elemental nature of thealloy and energy-dispersive X-ray spectroscopy established a 6 wt %phosphorus composition. The cathode was found to exhibit desirable HERactivity in water at neutral pH with a Faradaic efficiency of 99±2%.Moreover, the activity of the Co—P alloy surpassed the activity of theearth-abundant NiMoZn and stainless steel (SS) cathodes used in previousstudies, FIG. 14. At constant voltage, a stable HER current wasmaintained for at least 16 days, see FIG. 15. Negligible H₂O₂ wasproduced during HER as compared to stainless steel (SS) and platinum(Pt) cathodes, see FIG. 16.

FIG. 17 presents, cyclic voltammograms of Co²⁺ in the phosphate buffer(pH=7), a pre-wave to the catalytic water-splitting current correspondsto the oxidation of Co²⁺ to Co³⁺, which drives deposition of thecatalyst. The CoP_(i) catalyst is also known to exhibit a depositionrate that is linearly proportional to Co²⁺ concentration. As notedpreviously, the self-healing property of CoP_(i) alloy is derived fromthis interplay of the potential at which OER occurs vs. the potential atwhich the catalyst deposits. In concert, the Co—P and CoP_(i) catalystsmaintain extremely low concentrations of Co²⁺ in solution throughactivity derived from the self-healing process. Inductively coupledplasma mass spectrometry (ICP-MS) analysis of a Co-P|CoP_(i) catalystssystem (E_(appl)=2.2 V) confirm that sub-μM levels of Co²⁺ are presentin solution after 24 h. This concentration of Co²⁺ (0.32±0.06 μM) iswell below the concentration of Co²⁺ (half maximal inhibitoryconcentration, IC₅₀ of about 25 μM) that is toxic to R. eutropha asillustrated by the test results shown in FIG. 18. When diffusion betweenthe two electrodes is impeded by a porous glass frit, Co²⁺concentrations rose to about 50 μM. It is noted for a NiMoZn cathode,Ni²⁺ concentrations are not regulated by self-healing as NiP_(i) cannotform from P_(i), and the deposition to NiO_(x) occurs at >1.5 V vs. NHEas also shown in FIG. 17.

Example: Growth Under Nutrient Constraints

Metabolic engineering of R. eutropha enables the renewable production ofan array of fuels and chemical products. Specifically, when R. eutrophaconfronts nutrient constraints coupled with carbon excess, thebiosynthesis of poly(3-hydroxybutyrate) (PHB) is triggered in thewild-type H16 strain as an internal carbon storage, see FIG. 1B. Assuch, digestion is used for PHB collection. Under a constant rate ofwater splitting, PHB synthesis was not manifest until nitrogen becamelimiting at around 2 days, indicated by the cessation of biomassaccumulation, see FIG. 19, as well as the η_(elec) taken every 24 hoursshown in FIG. 20. Using a titer of about 700 mg/L, the 6-d average forPHB synthesis was measured to be η_(elec)=36±3%, see FIG. 6, with a 24-hmaximum of η_(elec)=42±2% (n=3) see FIG. 20. In engineered strains, thisPHB pathway can be modified to excrete the fusel alcohols isopropanol(C₃), isobutanol (C₄), and 3-methyl-1-butanol (C₅) with energy densitiesof 24, 28, and 31 MJ/L, respectively as well as other possible products.The culture supernatant was analyzed to quantify the secreted alcohols.The accumulation of these liquid fuels follows similar trends asobserved for PHB synthesis. FIGS. 21 and 23 show that biomass productionplateaus while isopropanol titers grow to about 600 mg/L and C₄+C₅alcohol titers grow to about 220 mg/L. Engineered R. eutropha strainproduced isopropanol with a 6-day average η_(elec)=31±4%, FIG. 6, with a24-hr maximum of η_(elec)=39±2% (n=4), FIG. 22. The bacterial strainengineered to produce C₄+C₅ alcohols averaged a 6-day η_(elec)=16±2%,FIG. 6, with a 24-hr maximum of η_(elec)=27±4% (n=3), FIG. 24. Theachieved titers were higher than previously reported values, andη_(elec) have been increased by at least 20 to 50 fold compared topreviously reported results.

FIG. 25 is a photograph of cell cultures taken for different isopropanolconcentration. As illustrated by the cultures, R. eutropha demonstratestolerance towards isopropanol, which allows for enriched productconcentrations under extended operation and is consistent with theobserved system behavior described above regarding continued productionand cell growth with increasing isopropanol concentrations.

Example: Reactive Oxygen Species (ROS) Resistance

A ROS-resistant variant of R. eutropha, evolved from a reactor includinga stainless steel CoP_(i) water-splitting electrode pair after 11consecutive days of operation at an applied voltage potential of 2.3 Vwith a H₂O₂ generation rate of ˜0.6 μM/min. Genome sequencing foundseveral mutations between the strain (BC4) and the wild-type (H16)described above in reference to Table I. In the presence of paraquat asa ROS-inducer, the IC₅₀ of paraquat for BC4 is almost one order ofmagnitude higher than that of wild type, see FIG. 26. However, there wasminimal difference noted in η_(elec) for the current reactor systemsusing the Co-P|CoP_(i) electrode pairs, confirming minimalconcentrations of ROS in the reactors. Of course, it should beunderstood that BC4 may be used in any reactor system, and may also bebeneficial for use in helping systems achieve a high η_(elec) where ROSare present in larger concentrations.

Example: Conversion Efficiencies

As noted above, the combined systems, catalysts, and bacteria describedherein, and illustrated in the experiments above, help to mitigatebiotoxicity at a systems level while also providing bacteria resistanceto the various toxicants present in the system, thus, allowingwater-splitting catalysis to be interfaced with engineered organisms torealize high CO₂ reduction efficiencies that exceed naturalphotosynthetic systems. Owing to low E_(appl) of 1.8-2.0 V for watersplitting, high η_(elec) are achieved that translate directly to highsolar-to-chemical efficiencies (η_(SCE)) when coupled to typicalsolar-to-electricity device (η_(SCE)=η_(solar)×η_(elec)). For aphotovoltaic device of η_(solar)=18%, the Co-P|CoP_(i) |R. eutrophahybrid system can achieve at least a η_(SCE)=9.7% for biomass, 7.6% forbio-plastic, and 7.1% for fusel alcohols. This approach allows for thedevelopment of artificial photosynthesis with efficiencies well beyondnatural photosynthesis, thus providing a platform for the distributedsolar production of chemicals.

While the present teachings have been described in conjunction withvarious embodiments and examples, it is not intended that the presentteachings be limited to such embodiments or examples. On the contrary,the present teachings encompass various alternatives, modifications, andequivalents, as will be appreciated by those of skill in the art.Accordingly, the foregoing description and drawings are by way ofexample only.

What is claimed is:
 1. A method comprising: splitting water using acathode including a cobalt-phosphorus alloy and an anode includingcobalt phosphate in a solution containing a chemolithoautotrophicbacteria to form hydrogen (H₂) and oxygen (O₂) in the solution;providing carbon dioxide (CO₂) in the solution; providing bioavailablenitrogen in the solution; maintaining the bioavailable nitrogen in thesolution to below a threshold nitrogen concentration to controlproduction of a product by the chemolitoautrophic bacteria.
 2. Themethod of claim 1, wherein the chemolithoautotrophic bacteria is aRalstonia eutropha bacteria.
 3. The method of claim 1, wherein thechemolithoautotrophic bacteria is resistant to reactive oxygen species.4. The method of claim 1, wherein the product is an alcohol.
 5. Themethod of claim 1, wherein the product is at least one of a fatty acid,an alkane, a polyhydroxyalkanoate, and an amino acid.
 6. The method ofclaim 1, wherein the solution includes a phosphate.
 7. The method ofclaim 1, further comprising continuously bubbling carbon dioxide throughthe solution.
 8. The method of claim 1, further comprising maintainingan isolated gas volume above a surface of the solution within a headspace of a reactor chamber.
 9. The method of claim 8, further comprisingreplenishing the isolated gas volume to an original composition at oneor more time intervals.
 10. The method of claim 8, wherein the isolatedgas volume comprises primarily carbon dioxide.
 11. The method of claim1, wherein splitting of the water is controlled to generate hydrogen ata rate equal to a rate of hydrogen consumption by the bacteria.
 12. Themethod of claim 1, wherein the solution is disposed in a reactorchamber.
 13. The method of claim 8, wherein maintaining the compositionof the isolated gas volume within the head space maintains theconcentration of the bioavailable nitrogen in the solution below thethreshold nitrogen concentration.
 14. The method of claim 1, furthercomprising sensing a concentration of one or more gases dissolved in thesolution, and controlling the concentration of the one or more gasesdissolved in the solution based at least in part on the sensedconcentration of the one or more gases dissolved in the solution. 15.The method of claim 14, further comprising changing the concentration ofthe one or more gases dissolved in the solution to be within apredetermined range based at least in part on the sensed concentrationof the one or more gases dissolved in the solution.
 16. The method ofclaim 14, wherein the one or more gases includes hydrogen, and furthercomprising changing a generation rate of hydrogen by the cathode and theanode to control the concentration of hydrogen in the solution.
 17. Themethod of claim 14, wherein the one or more gases dissolved in thesolution includes nitrogen gas dissolved in the solution.
 18. A methodcomprising: splitting water using a cathode including acobalt-phosphorus alloy and an anode including cobalt phosphate in asolution containing a chemolithoautotrophic bacteria to form hydrogen(H₂) and oxygen (O₂) in the solution, and wherein the solution includesbioavailable nitrogen.
 19. The method of claim 18, further comprisingproviding carbon dioxide (CO₂) in the solution.
 20. The method of claim18, further comprising continuously bubbling carbon dioxide through thesolution.
 21. The method of claim 18, further comprising maintaining thebioavailable nitrogen in the solution to below a threshold nitrogenconcentration to control production of a product by thechemolitoautrophic bacteria.
 22. The method of claim 18, wherein thechemolithoautotrophic bacteria is a Ralstonia eutropha bacteria.
 23. Themethod of claim 18, wherein the chemolithoautotrophic bacteria isresistant to reactive oxygen species.
 24. The method of claim 18,wherein the product is an alcohol.
 25. The method of claim 18, whereinthe product is at least one of a fatty acid, an alkane, apolyhydroxyalkanoate, and an amino acid.
 26. The method of claim 18,wherein the solution includes a phosphate.
 27. The method of claim 18,further comprising maintaining an isolated gas volume above a surface ofthe solution within a head space of a reactor chamber.
 28. The method ofclaim 27, further comprising replenishing the isolated gas volume to anoriginal composition at one or more time intervals.
 29. The method ofclaim 27, wherein the isolated gas volume comprises primarily carbondioxide.
 30. The method of claim 27, wherein maintaining the compositionof the isolated gas volume within the head space maintains theconcentration of the bioavailable nitrogen in the solution below athreshold nitrogen concentration.
 31. The method of claim 18, whereinsplitting of the water is controlled to generate hydrogen at a rateequal to a rate of hydrogen consumption by the bacteria.
 32. The methodof claim 18, wherein the solution is disposed in a reactor chamber. 33.The method of claim 18, further comprising sensing a concentration ofone or more gases dissolved in the solution, and controlling theconcentration of the one or more gases dissolved in the solution basedat least in part on the sensed concentration of the one or more gasesdissolved in the solution.
 34. The method of claim 33, furthercomprising changing the concentration of the one or more gases dissolvedin the solution to be within a predetermined range based at least inpart on the sensed concentration of the one or more gases dissolved inthe solution.
 35. The method of claim 33, wherein the one or more gasesincludes hydrogen, and further comprising changing a generation rate ofhydrogen by the cathode and the anode to control the concentration ofhydrogen in the solution.
 36. The method of claim 33, wherein the one ormore gases dissolved in the solution includes nitrogen gas dissolved inthe solution.